In this project, we propose to explore whether diet influences the development of cancer using a mouse model system. Our hypothesis is that diet exerts chronic and cumulative effects on gene regulation, so-called epigenetic effects, to cause a change from a normal pattern of regulation towards the pattern in early cancer formation. One key gene regulatory mechanism is the methylation of cytosines in the genome, usually associated with gene silencing. Cytosine methylation has been observed to be profoundly altered in a number of cancers, associated with gene expression changes that contribute to the cell's unregulated growth. The substrate for the methyl group added to cytosine are single carbon donor molecules like folic acid. It has been shown that altered folate levels in diet have substantial effects on overall levels of cytosine methylation in the genome, but the precise loci affected have not been identified. We developed a genome-wide assay to test ~600,000 sites in the mouse genome for cytosine methylation, allowing us to identify the loci affected in such situations. We propose a study in which we test normal cells from the jejunal area of the small intestine and adenomas from the same area, using mice that have been fed normal or single carbon donor-deficient diets. We will identify the epigenetic changes that occur (a) as a response to the diet deficient in folate, choline and methionine, and (b) in benign tumors from the same mice (using the Apc1638 model). We will define the degree of change in each case, a measure we call epigenomic distance, and determine whether diet-induced changes in normal cells represent an intermediate state on the way to neoplastic transformation by looking for consistency of the loci involved in each case. The successful outcome of this project will reveal insights into the early stages of cancer formation, link a specific type of dietary deficiency with aberrant methylation and cancer susceptibility, and create the basis for human studies for diagnostic and for prophylactic trials - it is conceivable that acquired epigenetic changes due to prolonged dietary influences could be reversed pharmacologically or through changed diet alone. This pilot project will serve as the foundation for an expanded project to address these possibilities. While diet is clearly influential in causing cancer of the gastrointestinal tract, the link between diet and the genetic changes required to cause a normal cell to become a cancer cell have remained elusive. In this study, we propose to use a mouse model system to test whether gene regulation is disturbed in intestinal tumours when exposed to a diet that influences one of the regulators of gene expression, cytosine methylation. The study will be the first genome-wide study of cytosine methylation to see whether diet-induced changes represent an intermediate stage in the development of cancer. [unreadable] [unreadable] [unreadable] [unreadable]